Secretome profiling of human epithelial cells exposed to cigarette smoke extract and their effect on human lung microvascular endothelial cells

Cigarette smoke (CS) is one of the leading causes of pulmonary diseases and can induce lung secretome alteration. CS exposure-induced damages to human pulmonary epithelial cells and microvascular endothelial cells have been extensively demonstrated; however, the effects of the secretome of lung epithelial cells exposed to CS extracts (CSE) on lung microvascular endothelial cells are not fully understood. In this study, we aimed to determine the effects of the secretome of lung epithelial cells exposed to CSE on lung microvascular endothelial cells. Human lung epithelial cells, A549, were exposed to CSE, and the secretome was collected. Human lung microvascular endothelial cells, HULEC-5a, were used to evaluate the effect of the secretome of A549 exposed to CSE. Secretome profile, endothelial cell death, inflammation, and permeability markers were determined. CSE altered the secretome expression of A549 cells, and secretome derived from CSE-exposed A549 cells caused respiratory endothelial cell death, inflammation, and moderately enhanced endothelial permeability. This study demonstrates the potential role of cellular interaction between endothelial and epithelial cells during exposure to CSE and provides novel therapeutic targets or beneficial biomarkers using secretome analysis for CSE-related respiratory diseases.

www.nature.com/scientificreports/ the secretion of endothelial cytokines, chemokines, and secretomes 13 .This highlights that the epithelium and endothelium may have crosstalk pathways and molecular connections during exposure to CS.However, the molecular mechanism underlying the effect of secretome derived from lung epithelial cells on lung endothelial cells has not been investigated in detail.This study aimed to determine the effects of CS extract (CSE) on lung epithelial cell secretome and its effects on lung microvascular endothelial cells and identify the secretome profile of human lung epithelial cells exposed to CSE.

The secretome of A549 cells exposed to CSE promotes inflammation and death of human lung microvascular endothelial cells
Annexin V/propidium iodide (PI) staining showed that the secretome group had a significantly higher percentage of early apoptosis (13.38 ± 0.2 vs. 5.12 ± 0.029, p < 0.05), late apoptosis (8.733 ± 0.116 vs. 1.633 ± 0.094, p < 0.05), and decreased cell viability (77.433 ± 0.068 vs. 91.767± 0.135, p < 0.05) than the control group (Fig. 3a,b).We performed quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis to determine the expression of the proinflammatory cytokine gene IL6, proinflammatory chemokine gene IL8, and Toll-like receptor genes TLR1, TLR2, and TLR4.The S group (cells cultured in the collected secretome) had a significantly higher level of IL8 than the control group (p < 0.05) and an increasing trend of IL-6 expression (Fig. 3f,g), while no significant changes in the expression of TLR1, TLR2, and TLR4 were noticed (Fig. 3c-e).These results indicate that the secretome of A549 cells exposed to CSE had a direct effect on inflammation and death of human respiratory microvascular endothelial cells.

The secretome of A549 exposed to CSE alters the integrity of human lung microvascular endothelial cells and the expression of caveolin-1 and claudin-5
Inflammation and death of respiratory endothelial cells can induce the dysfunction of the vascular permeability barrier.The integrity and permeability of human lung microvascular endothelial cells were evaluated by staining HULEC-5a cells with phalloidin-iFlour 555, caveolin-1, and claudin-5.HULEC-5a treated with the secretome of A549 cells exposed to CSE showed actin disorganization, loss of stress fiber, and altered morphology (Fig. 4a).In addition, we observed a moderate reduction of caveolin-1 and claudin-5 expression in the S group (Fig. 4b-d).These observations suggest that the secretome of respiratory epithelial cells can contribute to a weakened lung membrane integrity and may enhance lung microvascular permeability.

Analysis of the secretome of A549 cells exposed to CSE
The secretome data of human epithelial cells exposed to CSE identified a total of 2,894 proteins, among which 991 were identified to be unique in the secretome of the 10% CSE group; and 1,159 proteins were unique in the secretome of untreated A549 cells (Supplementary Materials; Table S1, Fig. 5a).Venn diagrams showed 278 upregulated proteins (p < 0.1) with respect to those of the control group (Fig. 5b).Ontological analysis of the identified proteins revealed their association with several functions, such as biological process involved in interspecies interaction between organisms (23.68%), immune system process (12.95%), protein localization (6.52%), and metabolic process (2.82%).Twenty top upregulated proteins were determined as shown in Table 1 and Fig. 5c.We identified von Willebrand factor A domain-containing protein 7 (VWA7) and Rho GTPase-activating protein 18 as crucial players in vascular biology, three proteins related to inflammation (Kelch-like protein 11, c-FOS, and transmembrane protein 187), two proteins involved in extracellular matrix remodeling (matrix remodelingassociated protein 5 and A disintegrin and metalloproteinase with thrombospondin motifs 20), and three proteins associated with actin and cytoskeleton [Ras-associated and pleckstrin homology domains-containing protein 1 (RAPH1), Capping protein, Arp2/3 and myosin-I linker protein 3, and MAP7 domain-containing protein 1).Furthermore, we identified three damage-associated molecular patterns (DAMPs), including decorin, heat shock protein-40, and beta-defensin, among 278 upregulated proteins.Further investigation showed that five dominant proteins (decorin, VWA7, c-FOS, beta-defensin, and RAPH1) are possibly involved in the extracellular matrix remodeling pathway, inflammation, cell death, and endothelium permeability pathway (Fig. 5d).

Discussion
CS is a major cause of human respiratory epithelial cell death, resulting in the secretion of several proteins, which finally leads to lung microvascular endothelial injury.The present study highlights that the secretome of human lung epithelial cells exposed to CSE can promote inflammation of human respiratory endothelial cells and moderately enhance vascular permeability.In addition, we showed the possible protein-protein interactions in this study.
CS contains several toxicants, such as nicotine, cotinine, 2-toluidine, and 2,6 dimethylaniline 3,4 .Previous preclinical and clinical studies have shown that CS can promote cell death, particularly in lung epithelial cells [14][15][16][17][18] .Consequently, several mechanisms underlying the effects of CS have been identified, including the direct promotion of oxidative stress, injury, and inflammation.Direct or indirect exposure to CS can disrupt the balance between oxidative stress and antioxidative systems by modulating glutathione, superoxide dismutase, and glutathione peroxidase activities 15 .Moreover, CS can directly induce superoxide anion generation, leading to oxidative stress-induced tissue damage 16 .Overproduction of reactive oxygen species triggers an intracellular signaling cascade that promotes pro-inflammatory mediator expression 17 .Oxidative stress and inflammation dysregulation are key active players that contribute to the activation of cell death 18 .Several studies have reported that CS significantly induces necrosis and alters the secretome of lung epithelial cells [19][20][21] .In the present study, human lung epithelial cells exposed to CS exhibited cytotoxicity and necrotic cell death in a dose-and timedependent manner, along with alteration in secretomic profile.Our results revealed varying percentages of cell death between the two methods (MTT and Annexin V/PI assay), reflecting the different levels of cellular mechanisms detected via both methods.Normally, alveolar epithelial and vascular endothelial cells form a semipermeable interface, which preserves the homeostasis of pulmonary microcirculation and maintains alveolar-capillary interaction 8,22 .Alteration of secretory proteins in the respiratory layer impairs its function.Several preclinical and clinical studies have reported that CS can alter respiratory secretome, leading to variation in epithelial-endothelial cell communication [10][11][12]22 . Thi secretory secretome can promote endothelial cell injury 10,21 .Although respiratory endothelial cell injury by direct CS exposure has been reported 2,7,21 , the molecular mechanism underlying the interaction between lung epithelial and endothelial cells during CS exposure has not been fully investigated, and the key role of endothelial cells during CS exposure is largely unexplored. The pesent study demonstrated, for the first time, the molecular mechanism of CS-exposed secretome derived from respiratory epithelial cells in pulmonary microvascular endothelial cells (Fig. 6).
Our findings showed that the CSE-exposed secretome significantly induced IL8 expression and reduced cell viability in the S group.These findings are consistent with those of a previous study 23 and suggest that secretome derived from CS-exposed lung epithelial cells can induce lung endothelial cell inflammation and cell death.IL-8, a proinflammatory chemokine and an autocrine/paracrine growth factor is a key player in neutrophil recruitment to the inflammation site and plays a crucial role in endothelial cell survival, proliferation, and angiogenesis 24,25 .Damaged respiratory endothelial cells secrete inflammatory mediators, which can further enhance inflammation, leading to endothelial cell death.Concordant with this, Demedts et al. have demonstrated lung endothelial cell death caused by CS-induced inflammation in patients 26 .These findings indicate a key role for lung endothelial cells in response to CS exposure.
The lung microvascular endothelial cell barrier forms tight and close contacts that regulate the movement of various molecules and leukocytes into the interstitium and subsequently into the respiratory air spaces 27 .A reduction in the integrity of lung microvascular endothelial cells results in increased endothelial permeability, www.nature.com/scientificreports/enhancing inflammation, inducing pulmonary edema, and progressing lung diseases 28 .Our results showed that lung endothelial cells treated with CS-exposed secretome derived from respiratory epithelial cells exhibited actin reorganization, moderately reduced caveolin-1 and claudin-5 expression and slightly decreased caveolin-1 and claudin-5 mRNA expression.These observations suggest that incubation for 6 h may be late for the detection of caveolin-1 and claudin-5 mRNA expression.However, under physiological conditions, the epithelial-endothelial membrane interaction is uninterrupted.Caveolin-1, a transmembrane protein, and claudin-5, a tight junction protein, both of which are abundant in lung endothelial cells, play important roles in regulating vascular permeability 29,30 .Cav1 −/− mutation can increase extravascular lung water by limiting water transport, thereby leading to pulmonary edema 31 , while overexpression of claudin-5 reverses oxygenation and modulates vascular leakage in a rat model of acute respiratory distress syndrome 32 .Therefore, the CS-exposed secretome of lung epithelial cells may increase vascular permeability.Several studies reported an increase in IL-8 expression in CSE exposure, which promotes protease enzyme activity and expression 23,33 .This increase in protease enzyme activity can lead to endothelial cell membrane destruction and loss of the actin cytoskeleton.However, the specific role of protease enzyme activity in endothelial permeability requires further investigation.Analysis of epithelial secretome revealed the five candidate proteins, including VWA7, c-FOS, beta-defensin, decorin, and RAPH1.Prediction of protein-protein interaction of secretome and endothelial receptors, particularly in Toll-like receptors (TLRs) 1, 2 and 4, demonstrated that these candidate proteins play important roles in several mechanisms of endothelial cells, especially those related to inflammation, cell death pathway, and actin reorganization.In this study, secretome profiles and candidate proteins of lung epithelial cells exposed to CSE were explored, revealing that the secretome can induce lung microvascular endothelial cell inflammation and increase vascular lung permeability.These results suggest a strong association between the predicted pathways and our previously mentioned results.These findings suggest novel targets for the treatment of CS-related respiratory diseases as well as potential biomarkers.However, the current study was performed only under 2D indirect co-culture conditions; therefore, further studies using an air-liquid interface system are required to support these findings.
Conclusively, the present study demonstrates the in vitro effects of secretome released from human alveolar epithelial cells exposed to CSE on human microvascular endothelial cells and reveals novel therapeutic targets and potential biomarkers.Nevertheless, the precise functions and underlying mechanisms of the identified candidate proteins and pathways in smoking-related diseases should be further validated in both preclinical and clinical studies.
Cells were maintained at 37 °C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Preparation and composition analysis of CSE
CSE was prepared using commercial cigarettes (Marlboro, Philip.Morris, Inc., Richmond, VA, USA), and mainstream smoke was bubbled through 1 mL/cigarette of RPMI-1640 medium using 50-mL glass syringes with glass plungers (50 mL puffs every 10 s), followed by pH adjustment (7.35-7.45).CSE was filtered through a 0.22-µM filter and used for further investigation.The CSE solution was considered to be 100%.To determine the chemical components of CSE, LC-ESI-QTOF 6540 (Agilent Technologies, Singapore) coupled with an Agilent 1260 infinity Series HPLC system (Agilent, Waldbronn, Germany) was used.The separation was performed with the Luna C18 column (4.6 × 150 mm, 5 µm; Phenomenex, USA) at a flow rate of 500 µL/min and control temperature of 35 °C.Water type I (Millipore, USA) with 0.1% (v/v) formic acid and acetonitrile B with 0.1% (v/v) formic acid were used as mobile phases A and B, respectively.The gradient elution mode started with 5% solvent B to 95% B linear gradient within 30 min and held on at this ratio for 10 min, followed by a 5-min post-run for column equilibrium.The injection volume was 5 µL.The operating parameters in MS detection were as follows: drying gas, N 2 ; flow rate, 10.0 L/min; drying gas temperature, 350 °C; nebulizer pressure, 30 psig; capillary, 3500 V; skimmer, 65 V; octapole RFV, 750 V; and fragmentor voltage, 100 V.The fragmentation MS/MS mode was set up at 3 collision energies of 10, 20, and 40 V using high-purity nitrogen gas (99.999%) as the collision gas.

Determination of cell viability and death
A549 cells were seeded in 96-well plates and cultured in the absence and presence of 5%, 10%, and 20% CSE at different time points (3, 6, 12, and 24 h).Cells were incubated with MTT solution (0.01 g/mL) for 2 h to measure cell viability.The formazan crystals were dissolved in dimethyl sulfoxide, and absorbance at 490 nm was measured using an EnSpire multimode plate reader (PerkinElmer).The optical density of the control group was considered

Figure 1 .
Figure 1.Analysis of the chemical composition of CSE using MS/MS.

Figure 2 .
Figure 2. Effects of CSE on A549 cell viability, cell death and cell morphology.The finding demonstrated CSE includes lung epithelial cell death in a dose-and time-dependent manner, and 10% CSE caused cell morphology alteration.(a) A549 cells were treated with CSE at different doses for various time periods.Cell viability was determined using an MTT assay.(b) A549 cell death evaluated using Annexin V/PI staining.(c) Percentage of cell death after exposure to 10% CSE.d.Control (a) and A549 cells were treated with 10% CSE for 3 h (b), 6 h (c), 12 h (d), 24 h (e), 36 h (f), and 48 h (g).

Figure 4 .
Figure 4. Effects of the secretome of A549 cells exposed to CSE on HULEC-5a cell integrity and viability.Secretomes derived from lung epithelial cells exposed to CSE reduce the integrity of the cells and increase the permeability of the respiratory endothelial cell.(a) Staining of actin cytoskeleton.(b) Immunofluorescence staining of caveolin-1 and claudin-5.(c) Caveolin-1 expression.(d) Claudin-5 expression.Data are presented as mean ± SEM. *p < 0.05 vs. the control group.

Figure 5 .
Figure 5.The secretome of A549 cells exposed to CSE and possible pathways of respiratory epithelialendothelial interaction during CSE exposure-correlation of inflammation mechanism, cell death pathway, and actin reorganization process.(a) Venn diagram; (b) volcano plot; (c) heatmap of top 20 proteins; (d) proteinprotein interaction by STRING analysis.

Table 1 .
Top twenty upregulated proteins in the secretome of A549 cells exposed to CSE.FC Fold change.

Table 2 .
Human primer sequences for RT-PCR.